Hepatocyte growth factor fragments that function as potent met receptor agonists and antagonists

a technology of hepatocyte growth factor and met receptor, which is applied in the field of polypeptide variants, can solve the problems of limited studies to develop met-targeted molecular imaging agents for non-invasive visualization of met expression in vivo compared to other cancer targets, and achieve the effects of promoting tissue repair and organ regeneration, promoting appropriate cell proliferation and migration, and reducing fibrosis

Active Publication Date: 2013-07-04
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]Alternatively, agonists of c-Met should promote tissue repair and organ regeneration in two ways: first, as a prophylactic, by protecting healthy cells from both necrotic and apoptotic death; and second, as a therapeutic, by promoting appropriate cell proliferation and migration needed for repair of pre-existing tissue injury. HGF agonists offer new therapeutic approaches to protecting major organs (including the liver, kidney, lung, heart, brain, and spinal cord) from injury, and reducing fibrosis in the liver, kidney, lung, and heart.

Problems solved by technology

There are currently no FDA approved therapeutics targeting the Met receptor, however, a few candidate molecules are in various stages of clinical trials.
In addition, studies to develop Met-targeted molecular imaging agents for non-invasive visualization of Met expression in vivo have been extremely limited compared to other cancer targets.

Method used

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  • Hepatocyte growth factor fragments that function as potent met receptor agonists and antagonists
  • Hepatocyte growth factor fragments that function as potent met receptor agonists and antagonists
  • Hepatocyte growth factor fragments that function as potent met receptor agonists and antagonists

Examples

Experimental program
Comparison scheme
Effect test

example 1

1.1 Protein Engineering of NK1 Through Yeast Surface Display

[0263]Yeast surface display is a powerful directed evolution technology that has been used to engineer proteins for enhanced binding affinity, proper folding, and improved stability. Combinatorial libraries of NK1 proteins were displayed on the surface of the yeast strain Saccharomyces cerevesiae through genetic fusion to the yeast mating agglutinin protein Aga2p. Aga2p is disulfide bonded to Aga1p, which is covalently linked to the yeast cell wall. In contrast to most yeast display studies, the construct we used here tethered the displayed NK1 proteins to the N-terminus of Aga2p (FIG. 2). It was found for this ligand-receptor system that this orientation reduced steric constraints of receptor and antibody labeling described below. The NK1 proteins were flanked by N-terminal hemagglutinin (HA) and C-terminal c-myc epitope tags, which were used to confirm expression of the construct on the yeast cell surface and to quantitat...

example 2

2.1 Soluble Production of Wild-Type NK1 and NK1 Mutants in the Yeast Strain P. Pastoris

[0275]Briefly, DNA encoding for wild-type NK1, M2.1, or M2.2 containing an N-terminal FLAG epitope tag (DYKDDDDK) and a C-terminal hexahistidine tag were cloned into the secretion plasmid pPIC9K. Constructs were transformed into P. pastoris, and were selected for growth on YPD-agar plates containing 4 mg / mL Geneticin and screened for NK1 expression by Western blotting of culture supernatant. FIG. 7A shows that M2.1 and M2.2 express well at 30° C., while wild-type NK1 expresses at much lower levels. This data is in agreement with previous studies that report engineering for enhanced protein stability using yeast-surface display also confers improved recombinant expression levels. However, reducing the expression temperature to 20° C. enabled efficient expression of wild-type NK1 (data not shown). NK1 and mutant expression were scaled up to 0.5 L in shake flask cultures and purified using immobiliz...

example 3

3.1 Point Mutations at the NK1 Homodimerization Interface

[0279]Residue N127 lies within the linker region connecting the N and K1 domains (FIG. 1). The side chain of this asparagine residue forms two hydrogen bonds. The N127D variant was frequently observed among the library-isolated variants. (Tables 2 and 3). To probe the effects of the N127D mutation within M2.2 on biological activity, a series of point mutants were generated at this position. An alanine residue transforms wild-type NK1 from an agonist into an antagonist by disrupting stabilizing interactions of the NK1 homodimer. The effects of mutations to lysine or arginine at this position were tested. These substitutions introduce steric and electrostatic obstructions through bulky, charged side-chains.

[0280]In addition, the point mutant D127N was analyzed; this reverts this position back to the wild-type asparagine residue. Within the context of M2.2, which contains the N127D mutation, these mutations are referred to as D12...

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Abstract

The NK1 fragment of hepatocyte growth factor (HGF) binds to and activates the Met receptor, a transmembrane receptor tyrosine kinase that plays a critical role in embryonic development and organ formation. The instant application discloses NK1 variant polypeptides which act as agonists or antagonists of HGF. Further disclosed are covalently linked NK1 variant polypeptides. Many of the disclosed variant polypeptides possess improved stability characteristics.

Description

PRIORITY CLAIM[0001]This application claims priority to U.S. provisional patent application Ser. No. 61 / 315,794, filed Mar. 19, 2010 and U.S. provisional patent application Ser. No. 61 / 411,080, filed Nov. 8, 2010, the contents of which are incorporated in their entirety.STATEMENT OF GOVERNMENT RIGHTS[0002]This invention was made in part with support from the United States Government (NIH NCI R21CA131706 and NIH NCI R01CA151706). The Government has rights in this invention.FIELD OF THE INVENTION[0003]The invention pertains to the field of polypeptide variants, in particular variants of Hepatocyte Growth Factor.BACKGROUND OF THE INVENTION[0004]Hepatocyte Growth Factor (HGF), also known as Scatter Factor (SF), is a multi-functional heterodimeric protein produced predominantly by mesenchymal cells and is an effector of cells expressing the Met tyrosine kinase receptor (“c-Met”)(Bottaro et al. (1991) SCIENCE 251: 802-804, Rubin et al. (1993) BIOCHIM. BIOPHYS. ACTA 1155: 357-371). Mature ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/475
CPCC07K14/4753A61K38/00
Inventor COCHRAN, JENNIFER R.JONES, II, DOUGLAS S.TSAI, PING-CHUAN
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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